Process for producing glutathione

ABSTRACT

There is disclosed a fermentation process for producing glutathione which comprises (a) the obtainment of a biomass pre-culture by pre-cultivating, in aerobic conditions, a strain of a glutathione producing yeast wherein the glutathione content per biomass unit is higher than 1.2% w/w; (b) the cultivation, in aerobic conditions, of the resulting biomass pre-culture such that the resulting biomass density is higher than 50 g/l; (c) the activation of the cultured biomass; and (d) the recovery of the cultured biomass, extracting glutathione at a pH equal to or lower than 6 and purifying the resulting glutathione. The process allows to obtain glutathione with high yields and relatively low costs.

[0001] The present invention relates to a process for producingglutathione. Particularly, the invention concerns a fermentation andactivation process for producing glutathione (hereinafter GSH) whichallows to obtain GSH with high yields and relatively low costs.

[0002] GSH, also known as N-(N-L-γ-glutamyl-L-cysteinyl)glycine, is aknown cellular tripeptide, present in virtually all cells. Its diversefunctions are important to many biomedical fields, including enzymology,transport, pharmacology, therapy, toxicology, endocrinology andmicrobiology, as well as to agriculture.

[0003] GSH plays a crucial role in various metabolic districts, as wellas in transport and in cellular protection. It serves the reduction ofthe disulfide linkages of proteins and other molecules, the synthesis ofthe deoxyribonucleotide precursors of DNA, and the protection of cellsagainst the dangerous effects of free radicals and of the many reactiveoxygen intermediates (e.g., peroxides) that are formed at various stagesof metabolism. The enzymatic and transport phenomena of GSH metabolismare treated in: Meister “Selective Modification Of GlutathioneMetabolism”, Science, Volume 220, No. 4596, 472-477 (1983).

[0004] Increasing the intracellular GSH levels, in various human bodydistricts, may be achieved by oral administration of pure GSH, or byinjection of certain GSH monoesters after dissolution in water (see, forexample, U.S. Pat. No. 4,710,489; U.S. Pat. No. 4,784,685 and U.S. Pat.No. 4,879,370).

[0005] GSH is produced industrially mainly by fermentation processeswherein the compound is extracted from microbial cells. Methods ofproducing GSH from microorganisms, are disclosed in various patents,including U.S. Pat. No. 4,596,775 and U.S. Pat. No. 4,582,801.

[0006] U.S. Pat. No. 4,582,801 discloses a process for producing GSHwith high yields (about 3-4% on dry cells and about 0.7-0.9% g/l),involving cultivatiting a strain belonging to the genus Saccharomyces,and having both an ability to produce GSH and a resistance to1,2,4-triazole or sodium azide, in a culture medium containingassimilable sources of carbon, nitrogen and inorganic salts,accumulating GSH in the microbial cells, harvesting the cells andrecovering GSH therefrom.

[0007] FR-A-2,692,280 discloses the use of zinc-resistant yeasts,belonging to the genus Saccharomyces, in a fermentation process forproducing GSH which is obtained in 4.1-6.6% on dry weight; the higherpercentage is obtained using L-cysteine which is known to ameliorate theaccumulation of GSH during the fermentation while negatively affectingthe production of the biomass.

[0008] Udeh K. O. and Achremowicz B., in “High-glutathione containingyeast Saccharomyces cerevisiae: optimization of production”, ActaMicrobiologica Polonica, 1997, vol. 46, No. 1, 105-114 debate about howto maximize GSH yield using a high-GSH containing yeast S. cerevisiaeS-8H and obtaining, a GSH average yield of 1.6 g/l and a GSH content of17 mg/g dry biomass.

[0009] Chi-Hsien Liu et al. in “Medium optimization for glutathioneproduction by Saccharomyces cerevisiae”, Process Biochemistry, vol. 34,1999, 17-23 report that glucose, peptone and magnesium sulphate aresuitable components for the cell growth and GSH production in the yeaststrain, the latter amounting, as an average, to 26-28 mg/l and to 124.93mg/l as a maximum.

[0010] Alfafara C. et al. in “Cysteine addition strategy for maximumglutathione production in fed-batch culture of Saccharomycescerevisiae”, Appl. Microbiol. Biotechnol., 1992, 37, 141-146 teach tomaintain a constant cysteine concentration in the reactor, during thefermentation, to maximize total GSH production amounting to about 0.8g/l.

[0011] Although these processes already represent an improvement for theproduction of GSH, they are sometimes either too complex or theirproduction yields are still relatively low, and a high production ofbiomass together with a specific productivity are still a goal for theproduction of GSH.

[0012] It has now been found that GSH can be produced by means of afermentation process which comprises:

[0013] (a) the obtainment of a biomass pre-culture by pre-cultivating,in aerobic conditions, a strain of a yeast genus selected fromSaccharomyces, Hansenula, Pichia, Candida, Cryptococcus,Schizosaccharomyces, Sporobolomyces, Bullera, Bulleromyces,Fiilobasidiella, Lipomyces, Rhodotorula or a stable mutant thereof,wherein the GSH content per biomass unit is higher than 1.2% w/w;

[0014] (b) the cultivation, in aerobic conditions, of the resultingbiomass pre-culture such that the density of the resulting culturedbiomass is higher than 50 g/l;

[0015] (c) the activation of the cultured biomass; and

[0016] (d) the recovery of GSH from the cultured biomass resulting fromstep (c) by extracting GSH, at a pH equal to or lower than 6, andpurifying the resulting GSH.

[0017] Particularly preferred as GSH producing yeasts are Pichiaangusta, identified in our strain collection as GN/2219, filed on Jul.13, 2000 with the “National Collection of Yeast Cultures” (NCYC),accession number: NCYC 2957; Saccharomyces cerevisiae, identified in ourstrain collection as GN/2220, filed on Jul. 13, 2000 with the NCYC,accession number: NCYC 2958; Saccharomyces cerevisiae var. ellipsoideus,identified in our strain collection as GN/2221, filed on Jul. 13, 2000with the NCYC, accession number: NCYC 2959; Candida boidinii, mutant 21,identified in our strain collection as GN/2222, filed on Oct. 24, 2000with the NCYC, accession number: NCYC 2983.

[0018] The yeast cells are either not mutagenized, or they can betreated with a mutagenic agent (e.g., UV light,1-methyl-3-nitro-1-nitrosoguanidine (NTG), methanesulfonic acid ethylester (EMS), etc.). The yeast cells which may be used for the mutantselection may show an oxidative stress response lower than the one ofthe original wild strain, in a pathway which is different from the GSHresponse pathway.

[0019]Pichia Angusta-NCYC 2957-GN/2219

[0020] The morphological, nutritional and sexual characteristics of thisstrain conform to those typical of this species, as described forinstance in: Kurtzman & Fell, “The Yeasts, a taxonomy study” (4^(th)edition) Elsevier, 1998.

[0021] In particular:

[0022] Morphological characteristics

[0023] In 5% malt extract, after 3 days of incubation at 25° C., thecells are spheroidal, single, in pairs or in small clusters.

[0024] In Dalmau plate agar, after 7 days of incubation at 25° C.neither pseudohyphae nor hyphae are observed under the coverglass.

[0025] At 25° C., in 2% malt extract or YM medium (3 g/l yeast extract;3 g/l malt extract; 5 g/l Bacto peptone—produced by Difco-; 10 g/lglucose; 20 g/l agar), ascospores are observed (ascospores arehat-shaped and usually non-conjugated). Fermentation Glucose +Trehalose + Galactose − Sucrose − Maltose − Lactose − Raffinose −Assimilation Sorbose (weak) + Mannitol + Sucrose + Glucitol + Maltose +α-methyl-D-glucoside + Trehalose + Salicin (weak) + Melezitose +Gluconate (weak) + Xylose + Succinate + Glycerol + Citrate +Erythritol + Nitrate + Ribitol + Vitamin-free medium −

[0026] Other characteristics: starch is not observed; gelatinliquefaction is very weak; maximum growth temperature is 48° C.

[0027]Saccharomyces cerevisiae-NCYC 2958-GN/2220

[0028] The morphological, sexual and nutritional characteristics of thisstrain conform to those typical of these species, as described forinstance in: J. A. Barnet et al.: YEAST: Characteristics andidentification. Third ed. Cambridge University, 2000, in particular:

[0029] Growth in “Malt extract”: after 3 days at 25° C. the cells areellipsoidal to cylindrical. A sediment, occasionally a ring, is present.

[0030] After one month at 20° C., a sediment is present.

[0031] Growth on “Malt agar”: after one month at 20° C., the streakculture is butyrous, cream to slightly brownish.

[0032] Formation of ascospores: the asci contain one to four ascospores.

[0033] Acetate agar is the best medium for inducing sporulation.Assimilation of carbon compounds Glucose + Dl-Lactate − Galactose −Glycerol − Ribose − Sorbitol − Saccharose + Erythritol − Maltose +Mannitol − Cellobiose − Inositol − Lactose − Sorbose − Melibiose −Glucuronate − Raffinose + Gluconate − Trehalose − Glucosamine −L-Arabinose − N-acetyl− − glucosamine D-xylose − α-methyl-D− − glucosideRamnose − 2-Ketogluconate − Levulinate − Palatinose + Melezitose −Esculine − Actidione − Fermentation 3 days 24 days Cellobiose − −Galactose − − Glucose + + Lactose − − Maltose + + Melibiose − −Sucrose + + Trehalose − −

[0034]Saccharomyces cerevisiae var. ellipsoideus-NCYC 2959-GN/2221

[0035] The characteristics of this strain are identical to the onesreported above for Saccharomyces cerevisiae-NCYC 2958-, GN/2220,excepted both the assimilation of carbon compounds and the fermentationtests. Assimilation of carbon compounds Glucose + Dl-Lactate −Galactose + Glycerol − Ribose − Sorbitol − Saccharose + Erythritol −Maltose + Mannitol − Cellobiose − Inositol − Lactose − Sorbose −Melibiose − Glucuronate − Raffinose + Gluconate − Trehalose −Glucosamine − L-Arabinose − N-acetyl− − glucosamine D-xylose −α-methyl-D- − glucoside Ramnose − 2-Ketogluconate − Levulinate −Palatinose + Melezitose − Esculine − Actidione − Fermentation 3 days 24days Cellobiose − − Galactose − + Glucose + + Lactose − − Maltose + +Melibiose − − Sucrose + + Trehalose − −

[0036]Candida boidinii (mutant 21)-NCYC 2983-GN/2222

[0037] The morphological, sexual and nutritional characteristics of thisstrain conform to those typical of these species, as described forinstance in: J. A. Barnet et al.: YEAST: Characteristics andidentification. Third ed. Cambridge University, 2000, in particular:

[0038] Growth in glucose-yeast extract-peptone water: after 3 days at25° C. the cells are long-ovoid to cylindrical, after slightly curved.

[0039] Growth on glucose-yeast extract-peptone agar: after one month at25° C. the streak culture is yellowish cream-coloured, soft, delicatelywrinkled.

[0040] Dalmau plate culture on corn meal agar: the pseudomycelium ispresent and with short ramified hyphae which bear verticillated chainsand groups of ovoidal blastospores. Assimilation of carbon compoundsGlucose + Dl-Lactate + Galactose − Glycerol + Ribose + Sorbitol +Saccarose − Erythritol + Maltose − Mannitol + Cellobiose − Inositol −Lactose − Sorbose − Melibiose − Glucuronate − Raffinose − Gluconate −Trehalose − Glucosamine − L-Arabinose − N-acetyl +− glucosamineD-xylose + α-methyl-D- − glucoside Ramnose − 2-Ketogluconate −Levulinate − Palatinose −+ Melezitose − Esculine − Actidione +Fermentation 3 days 24 days Cellobiose − − Galactose − − Glucose + +Lactose − − Maltose − − Melibiose − − Sucrose − − Trehalose − −

[0041] The process of the invention provides an economic way forproducing GSH, allowing to improve the productivity of the GSH producingyeasts as well as the rapid isolation of GSH overproducing mutants, theinexpensive cultivation of such mutants, the increase the GSH content ofthe biomass and the rapid and effective extraction of GSH from saidbiomass.

[0042] The cells of the yeast suitable for carrying out the process ofthe invention are haploid.

[0043] Either steps (a) and/or (b) and/or (c) of the process of theinvention can be carried out in a nutrient aqueous, liquid or solid,medium comprising at least one of the following compounds:

[0044] (i) a compound of a metal selected from Cd, V, Cu, Fe, Pb, Al,Co, Cr, Mn, Ni, Mo, Hg;

[0045] (ii) a peroxyde (e.g., H₂O₂, t-butyl peroxide, etc.);

[0046] (iii) an aldehyde (e.g., methylglyoxal, formaldehyde,acetaldehyde, etc.);

[0047] (iv) a hydroperoxyde (e.g., t-butyl-HOOH, etc.);

[0048] (v) a fatty acid (e.g., oleic acid, linolenic acid, arachidonicacid, etc.) and/or a linear or branched, saturated or unsaturated,derivative thereof;

[0049] said medium further comprising at least an assimilable source ofcarbon and/or nitrogen and/or at least a mineral salt, whenever thecompounds (i) to (v) are not such a source and/or salt themselves.

[0050] The sources of carbon can come, for instance, from agriculturaland/or industrial wastes; particularly, they can comprise at least oneof the following substances: sugars, such as dextrose, dextrin, glucose,fructose, saccharose, mannitol, mannose; organic acids; alcohols;aldehydes; glycerol; starch; fats; oils; hydrocarbons and whey and thelike; particularly, the production of GSH in accordance with theinvention can be carried out under favourable conditions employingrelatively non-expensive carbon sources, such as beet molasses.

[0051] The nitrogen sources can comprise, for instance, at least one ofthe following substances: malt extract, corn steep liquor, enzymatichydrolysate of casein, soya flour, dry yeast, peptone, soy peptone, meatextract, nitrate, amino acids, casein, ammonium salts and the like. Goodresults are obtained using ammonium salts such as ammonium nitrate andammonium sulphate.

[0052] The mineral salts used for the production can vary depending onthe culture medium; the soluble inorganic salts which provide sodium,potassium, magnesium, sulphate, chloride, nitrate ions, can be used.Such salts can be, for instance, monobasic potassium phosphate,magnesium sulphate, monobasic potassium sulphate, sodium nitrate. Theaddition of calcium carbonate may also be useful.

[0053] Further, the medium may comprise at least an amino acid (e.g.cysteine, methionine, glutamate, glutamine, glycine, leucine,acetylcysteine etc.) and/or a phosphorus source (e.g., potassiumphosphate, etc.) and/or an alcohol (methanol, ethanol, isopropanol,butanol, etc.)

[0054] Preferably, in step (a), the GSH content per biomass unit ishigher than 1.6% w/w whereas step (b) is preferably carried out at20-50° C. for 12-72 h, particularly at 25-45° C. for 12-48 h; thedensity of the cultured biomass being preferably between 50 and 65 g/l.

[0055] The process of the invention and, specifically, either steps (a)and/or (b) and/or (c), may be carried out either batch-wise orcontinuously.

[0056] Steps (a) and/or (b) and/or (c) of the process of the inventioncan be carried out by aeration with either air or oxygen gas and/or amixture thereof; the process of the invention is advantageously carriedout in aerated fermenters in order to get remarkable amounts of GSH. Thepreferred fermenter is an aeration agitation-type or an air-liftfermenter. Further, the process can be carried out in a flask and infermenters of different capacity.

[0057] The process of the invention encompasses the activation-step(c)—of the cultured biomass. In the present specification, “activation”is meant to indicate an enrichment of the intracellular GSH content ofthe biomass carried out employing resting cells in non-growingconditions.

[0058] Advantageously, the activation comprises:

[0059] (α) resuspending the cultured biomass, for instance, 5-20% of thedry biomass, in a water solution containing a 0.4-1 M carbon source;

[0060] (β) stirring the resulting suspension at 300-600 rpm; and

[0061] (γ) aerating said suspension with either air or oxygen gas and/ora mixture thereof, preferably at 1-4 vvm.

[0062] Preferably, in step (a), the water solution contains 0.001-0.01 Mcysteine, glycine and glutamate.

[0063] In another preferred embodiment, GSH, in step (d) of the processof the invention, is extracted by lysis at a pH of 0.5-3.0 and at atemperature of 70-90° C.

[0064] The cultivated biomass resulting from step (b) shows aproductivity of GSH from about 0.9-1.2%-whereas the activated culturedbiomass resulting from step (c) of the process of the invention shows anincrease of the GSH productivity to 2.7-3.9% under dried conditions.

[0065] The extraction of GSH in step (d) can be advantageously carriedout through a strong cationic resin such as, for instance, Amberlite® IR120-200-220 produced by Rhom & Haas or Relite® CF produced by MitsubishiChemical Corporation and, subsequently, a non-ionic resin such as, forinstance, SP 207 (Resindion, Mitsubishi). The GSH extraction can bepreferably obtained through precipitation of the salt, in the presenceof H₂SO₄ in which the salt, after salification of a H₂SO₄ becomesinsoluble.

[0066] The following examples illustrate the invention without limitingit.

EXAMPLE 1

[0067] One wild strain of Pichia angusta isolated from soil samples wasused, namely a homothallic haploid.

[0068] Stage 1

[0069] The strain was cultured for 20 hours at 37° C. (250 ml conicalflasks, 200 rpm orbital incubation) in 100 ml Rich Medium (RM) havingthe following composition (g/l): Glucose 20 Peptone 20 Yeast Extract 10

[0070] Stage 2

[0071]25 ml of yeast culture were spin-washed in distilled water twiceand re-suspended in distilled water.

[0072] 5×10⁷ cells were plated out onto Glucose Mineral Medium (GM)containing 8 mM cadmium chloride monohydrate (MMC) and having thefollowing composition (g/l): Glucose 20 Yeast Nitrogen Base 7 Cadmiumchloride monohydrate 1.61

[0073] The plates were UV irradiated to 90% mortality and incubated at37° C. in the dark, for 20 days.

[0074] Stage 3

[0075] After incubation, a typical plate showed 800-1000 colonies, about50% of which had a diameter F<1 mm (“small”) and about 50% had F>1 mm(“large”). Ten large colonies were isolated, streak purified onto RMplates, and re-tested onto MMC. Five out of ten colonies confirmed theircadmium-resistant phenotype.

[0076] Stage 4

[0077] Five colonies isolated in stage 3 were treated as described aboveyet, in this case, the cadmium was replaced with 200 mM of sodiumorthovanadate. At the end, five colonies were isolated confirming theircadmium-vanadate-resistant phenotype.

[0078] Stage 5

[0079] Each of the five cadmium-vanadate-resistant colonies of theprevious stage (hereinafter CVCdA, CVCdB, CVC, CVD, CVE) werepre-cultured in 7.5 ml Mineral Medium (MM) and incubated 16 h at 37° C.(orbital, 200 rpm). Cells from these pre-cultures were then used toinoculate 100 ml flasks containing 20 ml MM, so as to start with 5×10⁶cells/ml. The flasks were incubated at 37° C. for 24 h (orbital, 200rpm); the experiment was done in triplicate.

[0080] Stage 6

[0081] The cell content of each flask was collected by centrifugation,washed twice, and divided into two aliquots. One aliquot served todetermine the dry weight of the biomass, whereas the other served todetermine the GSH content of cell extracts, obtained by re-suspendingthe cell in 1 ml perchloric acid, adding 0.5 g glass beads (Sigma #G-9268), stirring 1 min×3, precipitating the solid phase bycentrifugation, and pipetting on the extract, according to the method ofAkerboom and Sies “Methods in enzymology”, vol. 27, pp.373-384, 1981,Academic Press Inc.

[0082] Table 1 shows some significant results obtained with the fivecultures, expressed as percentage of the weight levels per unit biomass.TABLE 1 GSH/biomass unit Strain E. 1 E. 2 E. 3 Mean Wild type 100 100100 100 (control) Mutant CVCdA 124 119 122 122 Mutant CVCdB 231 219 242231 Mutant CVC 100 101 97 99 Mutant CVD 102 105 106 104 Mutant CVE 109110 107 109

[0083] It will be noted that mutants CVCdA and, remarkably, CVCdB(Pichia angusta, identified in our strain collection as GN/2219, NCYC2957) show a significant increase in the intracellular GSH content.

[0084] Stage 7

[0085] The production of GSH using mutant CVCdB GN/2219, NCYC 2957indicated in Table 2 as mutant B, in a 3 l fermenter was carried outemploying two different media (GM, GlyM—as GM, yet glucose is replacedby 2% glycerol—and mM—as GM, yet glucose is replaced by 18% molasses,corresponding to 9% as saccharose), and the following cultureconditions: volume: 2l, temperature: 35° C., air: 1 vvm, pH: 5.0.

[0086] The evolution of GSH and biomass was followed for 62 h, andcompared to that of an identical control culture (wild strain). Theresults are shown in Table 2. TABLE 2 Biomass evolution GSH evolution(dry weight) g/dl Mg/g (dry weight) Wild type Wild type (control MutantB (control) Mutant B Medium GM Gly M mM GM Gly M mM GM Gly M mM GM Gly MmM Start 0.002 0.002 0.002 0.002 0.002 0.002 — — — — — — 14 h 0.4 0.412.3 0.4 0.4 12.1 0.9 0.8 0.8 2.1 2.3 2.4 24 h 4.1 4.5 37.4 4.2 4.3 37.79.8 9.7 9.7 17.8 16.8 17.1 38 h 5.3 5.7 54.2 5.4 5.5 53.3 7.8 7.9 7.816.1 17.1 17.2 41 h 6.4 6.6 55.1 6.7 6.7 55.4 4.5 4.3 4.6 8.4 7.9 15.962 h 12.7 12.6 59.7 12.9 12.7 59.5 2.7 3.1 3.2 5.1 5.4 13.3

[0087] It will be noted that the cellular productivity is higher in thecase of the Mutant B than in the wild strain, whereas the yielddifferences between the two media are not relevant.

[0088] These tests show that the process of the invention can be carriedout under favourable conditions employing various media and for longperiods of time.

[0089] After the fermentation phase, the biomass was recovered, washedand then either re-suspended in a concentrated form to start anactivation stage, aiming at furtherly increase GSH yields (described inexample 2), or processed to extract and purify GSH (herebelow describedin stage 8).

[0090] Stage 8

[0091] The biomass obtained from the previous stage was re-suspended indemineralised water to a concentration of 7 g/l (biomass dry weight). 10litres of this concentrated biomass suspension (CoBS) were permeabilisedusing a combination of low pH (1-6.5, with concentrated H₂SO₄) and hightemperature (75° C.×3 min). The biomass was then removed by filtration(0.2 mm ceramic membranes), and the GSH containing solution wasconcentrated by reverse osmosis so as to reach about 6 g/l GSH.

[0092] Tab. 3 summarizes the GSH yields (%) obtained at various pH,using the aforementioned CoBS. TABLE 3 pH 6.5 5 4 3 1.5 E. 1 0.50 0.550.61 0.68 0.98 E. 2 0.48 0.52 0.58 0.72 1.02 E. 3 0.42 0.49 0.64 0.730.95

[0093] These tests show that, under the conditions employed, cellpermeabilisation is favoured by low pHs.

[0094] Stage 9

[0095] The purification of GSH was achieved by percolating 1 l of theconcentrated GSH solution (about 6 g/l) in a column packed withAmberlite (1 l, IR120H), at a flow rate of 1 BV/h. 30 BV ofdemineralised water are then applied to wash the resin, before elutingthe GSH with 1% H₂SO₄. 6 l of the resulting eluates are concentrated to5 l by reverse osmosis, yielding about 85% of the initial GSH.

[0096] Stage 10

[0097] The concentrated eluates were percolated in a column packed with1 l of non-ionic porous resin i.e. SP207 (Resindion, Mitsubishi),previously regenerated at 1 BV/h. GSH was then eluted with water, andfractions 2 to 9 were collected. The GSH aqueous solution thus obtainedwas concentrated to 500 g/l. GSH was finally precipitated by a 50%ethanol solution. 4 g of white crystalline powder was obtained, with 98%GSH content.

EXAMPLE 2

[0098] A non sporulating spontaneous mutant strain of Saccharomycescerevisiae (identified in our strain collection as GN/2220, NCYC 2958),having a GSH content per biomass unit higher than 1.4% w/w, was used.

[0099] Stage 1

[0100] The strain was batch cultured for 24 h in a 14 l fermenteremploying three different media, namely (amounts are expressed as g/l)YP (20 peptone, 10 yeast extract, 10 glycerol, pH 5), YPG (20 peptone,10 yeast extract, 20 glucose, pH 5) and B (60 beet molasses—equivalentto 30 sucrose-, 5 (NH₄)₂SO₄, 1 K₂HPO₄, 0.5 yeast extract, 0.2 MgSO₄, pH5.8). Cultivation conditions were as follows: 28° C., 1 vvm air, 400rpm. Antifoam: Sigma PPG #2000 Inoculum 10% (V/V) of a stationary-phaseflask culture in YP.

[0101] Table 4 summarizes the results of seven different batches. TABLE4 Biomass GSH Y1 P Y1 Y2 P E. M g/l g/lh mg/l % mg/lh 1 YPG 9.40 0.39 94 1.0 3.90 2 YPG 9.20 0.39  65 0.7 2.70 3 B 10.7 0.44 207 1.9 8.60 4 B9.10 0.38 155 1.7 6.50 5 B 10.5 0.44 203 1.9 8.46 6 YP 7.50 0.31  90 1.23.80 7 YP 7.80 0.32 101 1.2 4.00

[0102] It will be noted that the cellular productivity, in the case ofthe medium B is more than double than in the case of the other twomedia; also the relative final yields, which differ considerably amongthe three media, show that B allows to obtain the best results.

[0103] Stage 2

[0104] Biomass obtained from the previous stage was collected bycentrifugation at 10,000 rpm for 10 min (5° C.), washed twice withdeionized water and re-suspended to 7% concentration (dry biomass, w/v)in the following solution (g/l): Glucose 82 Sodium tartrate 10 Adenine 4Cysteine 4 Methionine 3.5 KH₂PO₄ 3.5

[0105] The “biomass activation” process was then started in a 3 lfermenter for 24 h, with the following operating conditions: agitationspeed, 500 rpm; aeration rate, 3 vvm; temperature, 28° C.

[0106] Table 5 summarizes the GSH yields of four activation experiments,employing biomass obtained either in B medium, or in YPG medium. TABLE 5GSH Yield (% of dry biomass) Fermentation Before After E. mediumactivation activation 1 YPG 1.0 2.0 2 YPG 0.7 2.0 3 B 1.9 3.7 4 B 1.73.4

[0107] It will be noted that, independently of the initial GSH content,the aforementioned 24 h activation process allows significant GSH yieldincreases.

EXAMPLE 3

[0108] The strain of Saccharomyces cerevisiae NCYC 2958 was batchcultured for 30 h in a 50 l fermenter using the medium B (example 2).Inoculum 20% of stationary-phase. Cultivation conditions were as in theexample 2.

[0109] During the growth, starting form the 12th h, five additions(every 3 h) of molasses were done, so that the concentration ofsaccharose was kept higher than 10% until the 24th h.

[0110] A biomass of 58, 89 g/l and 1, 45% of GSH both on dry weight wereobtained.

[0111] The biomass collected for centrifugation (example 2), wassuspended again at 10% and “activated” in the following solution (g/l):Glucose 82 Sodium tartrate 10 Adenine 4 Cysteine 4 Sodium glutamate 5Glycine 5 Methionine 3 85% H₃PO₄ 0.25 ml

[0112] At the same conditions of example 2, 3.7% GSH was obtained underdry conditions, equal to 3.7 g/l of “activation”.

EXAMPLE 4

[0113] One wild strain of Candida boidinii, isolated from plantmaterial, was mutagenised, and mutant #21 was selected following aprocedure analogous to the one described in Example 1, but usingmethylglioxal as a selective agent. This strain was identified asCandida boidinii mutant 21, NCYC 2983, internal strain collectionGN/2222, and showed a GSH content per biomass unit higher than 1,5% w/w.

[0114] Stage 1

[0115] The strain was batch cultured for 24 h in a 14 l fermenteremploying medium B, but the molasses were added until 180 g/l (90 g/l assaccharose) as in example 3). Cultivation conditions were as follows:28° C., 1 vvm air, 400 rpm. Antifoam: Sigma PPG # Inoculum 10% (v/v) ofa stationary-phase flask culture in YP.

[0116] Table 6 summarizes the results of the fermentation. TABLE 6Biomass GSH Y1 P Y1 Y2 P H g/l g/lh mg/l % mg/lh  6 0.54 0.09 2.7 0.50.33 18 16.2 0.9 194.4 1.2 10.80 24 29.2 1.2 408.8 1.4 17.03 30 36.1 1.2469.3 1.3 15.64

[0117] Stage 2

[0118] The biomass obtained from the aforementioned batch was treated asdescribed in Example 1 (stage 8) in order to obtain a concentrated GSHsolution (about 6 g/l). 1 l of this solution was adjusted to pH 1.8 byH₂SO₄, and then treated with a 10 ml suspension containing 1.4 g of Cu₂Oin water. The suspension was dropped very gently while the temperaturewas maintained at 4° C. The solution was then gently stirred at 4° C.for 2 h, and then undisturbed overnight.

[0119] Stage 3

[0120] After filtering, the precipitate (9 g) was washed several timeswith diluted H₂SO₄ and then solubilised by H₂S at room temperature. Thesolution, containing 4.5 g of GSH was applied to 1 l of SP 207, a nonionic resin produced by Mitsubishi Chemical, and then eluted with 10 BVof demineralised water. Fractions were collected (6 l), concentrated andtreated with 50% ethanol in water to precipitate GSH. 3.5 g of GSH,showing a 99% purity, were obtained.

EXAMPLE 5

[0121] The biomass obtained according to the examples 1 and 4 wastreated as described in the example 1 (stage 8) in order to obtain aconcentrated solution of GSH (about 6 g/l). 1 l of this solution wasadjusted at pH 1.8 by H₂SO₄ and heated at 40° C. in waterbath.

[0122] At a temperature of 40° C., 10 ml of a Cu₂O suspension (1.5 g)were added into water.

[0123] After 3 minutes, the resulting solution was quickly brought at 4°C. and then kept under agitation for 2 hours.

[0124] After 2 hours, it was proceeded to filtration as in the example 3(stage 3). After filtration, the precipitate (0.5 g) was dissolved inH₂S, purified with SP 207, a non ionic resin produced by MitsubishiChemical, and precipitated in ethanol. 3.6 g of GSH, having a 99%purity, were obtained.

1. A fermentation process for producing glutathione which comprises: (a) the obtainment of a biomass pre-culture by pre-cultivating, in aerobic conditions, a strain of a yeast genus selected from Saccharomyces, Hansenula, Pichia, Candida, Cryptococcus, Schizosaccharomyces, Sporobolomyces, Bullera, Bulleromyces, Cryptococcus, Filobasidiella, Lipomyces, Rhodotorula or a stable mutant thereof, wherein the glutathione content per biomass unit is higher than 1.2% w/w; (b) the cultivation, in aerobic conditions, of the resulting biomass pre-culture such that the density of the resulting cultured biomass is higher than 50 g/l; (c) the activation of the cultured biomass; and (d) the recovery of glutathione from the cultured biomass resulting from step (c) by extracting glutathione, at a pH equal to or lower than 6, and purifying the resulting glutathione.
 2. Process according to claim 1, wherein the strain is Pichia angusta-NCYC 2957-(GN/2219).
 3. Process according to claim 1, wherein the strain is Saccharomyces cerevisiae-NCYC 2958-(GN/2220).
 4. Process according to claim 1, wherein the strain is Saccharomyces cerevisiae var. ellipsoideus-NCYC 2959-(GN/2221).
 5. Process according to claim 1, wherein the strain is Candida boidinii, mutant 21-NCYC 2983-GN/2222).
 6. Process according to any of the previous claims, wherein either steps (a) and/or (b) and/or (c) are carried out in a nutrient aqueous, liquid or solid, medium comprising at least one of the following compounds: (i) a compound of a metal selected from Cd, V, Cu, Fe, Pb, Al, Co, Cr, Mn, Ni, Mo, Hg; (ii) a peroxyde; (iii) an aldehyde; (iv) a hydroperoxyde; (v) a fatty acid and/or a linear or branched, saturated or unsaturated, derivative thereof; said medium further comprising at least an assimilable source of carbon and/or nitrogen and/or at least a mineral salt, whenever the compounds (i) to (v) are not such a source and/or salt themselves.
 7. Process according to the previous claim, wherein the compound (i) is a mineral inorganic water soluble salt.
 8. Process according to claim 6 or 7, wherein the medium comprises at least an amino acid and/or a phosphorus source and/or an alcohol.
 9. Process according to any of the previous claims, wherein, in step (a), the glutathione content per biomass unit is higher than 1.6% w/w.
 10. Process according to any of the previous claims, wherein step (b) is carried out at 20-50° C. for 12-72 h and the density of the resulting cultured biomass is between 50 and 65 g/l.
 11. Process according to any of the previous claims, wherein step (b) is carried out at 25-45° C. for 12-48 h.
 12. Process according to any of the previous claims, wherein either steps (a) and/or (b) and/or (c) are carried out either batch-wise or continuously.
 13. Process according to any of claims 6 to 12, wherein the carbon source comes from agricultural and/or industrial wastes.
 14. Process according to the previous claim, wherein the carbon source comprises at least one of the following substances: sugars, organic acids, alcohols, aldehydes, glycerol, fats, oils, hydrocarbons and whey.
 15. Process according to the claim 13 or 14, wherein the carbon source comprises beet molasses.
 16. Process according to any of claims 6 to 12, wherein the nitrogen source comprises at least one of the following substances: malt extract, corn steep liquor, enzymatic hydrolysate of casein, soya flour, dry yeast, peptone, soy peptone, meat extract, nitrate, amino acids, casein and ammonium salts.
 17. Process according to the previous claim, wherein the nitrogen source comprises ammonium nitrate or ammonium sulphate.
 18. Process according to any of the previous claims, wherein either steps (a) and/or (b) and/or (c) are carried out by aeration with either air or oxygen gas and/or a mixture thereof.
 19. Process according to any of the previous claims, wherein the activation comprises: (α) resuspending the cultured biomass (5-20% dry biomass) in a water solution containing a 0.4-1 M carbon source; (β) stirring the resulting suspension at 300-600 rpm; and (γ) aerating said suspension with either air or oxygen gas and/or a mixture thereof.
 20. Process according to the previous claim, wherein, in step (α), the water solution contains 0.001-0.01 M cysteine, glycine and glutamate.
 21. Process according to any of the previous claims, wherein the fermenter to be used is an aeration-agitation or an air-lift fermenter.
 22. Process according to any of the previous claims, wherein the glutathione in step (d) is extracted by lysis at a pH of 0.5-3.0 and at a temperature of 70-90° C.
 23. Process according to any of the previous claims, wherein the glutathione in step (d) is extracted through a strong cationic resin and, subsequently, a non-ionic resin. 